Hemoglobin (Hb) based blood substitutes are being developed rapidly to overcome the shortage of blood supply (Chang, 1999; Klein, 2000). The most extensively studied and financed blood substitute was diaspirin cross-linked Hb (Winslow, 2000a). Although intramolecular crosslinking of Hb overcame the nephrotoxicity and high oxygen affinity of the acellular Hb (Chang, 1999), the product remained vasoactive (Kramer, 2003; Winslow, 20006), which has been attributed to the scavenging of nitric oxide by the extravasated acellular Hb (Winslow, 2000a). Enhancing the molecular size of Hb by oligomerization and lowering the affinity of Hb to nitric oxide by site directed mutagenesis are two solutions to overcoming the vasoactivity of Hb. Animal studies have shown that both approaches reduce the pressor effect of Hb (Gulati et al., 1999).
Enzon PEGylated bovine Hb carries ten copies of polyethylene glycol 5000 (PEG-5K) chains, and was non-hypertensive (Conover et al., 1999). Its enhanced molecular volume, high viscosity and high colloidal osmotic pressure (COP) have been attributed as the molecular basis of neutralizing the vasoactivity of acellular Hb (Intaglietta, 1997). Accordingly, PEGylation of Hb has been considered as a new approach to generate non-hypertensive Hb (Rohlfs et al., 1998; Winslow et al., 1998). In an attempt to establish that the neutralization of the vasoactivity is a generalized consequence of PEGylation of Hb, a non-hypertensive hexaPEGylated Hb, (SP-PEG5K)6-Hb was generated using extension arm facilitated PEGylation protocol (Acharya et al., 2005; Manjula et al., 2005). Compared with the Enzon decaPEGylated bovine Hb, (SP-PEG5K)6-Hb has less number of PEG-5K chains conjugated, and the positive charge of Hb was not changed upon linking the extension arm or on conjugating the PEG-chains through the thiol groups at the distal end of the extension arms (conservative PEGylation) (Acharya et al., 2005; Manjula et al., 2005). Therefore, the results reflect the higher efficiency of conservative PEGylation to neutralize the vasoactivity of Hb.
The non-hypertensive PEGylated Hb (SP-PEG5K)6-Hb exhibits a very high oxygen affinity, which was considered as a consequence of the PEGylation at Cys-93(β) of Hb (Acharya et al., 2005). High oxygen affinity for an Hb-based oxygen carrier has been advocated as a desirable property to generate non-hypertensive Hb, as this will reduce the propensity of the acellular Hb to off load the oxygen on the arterial side of the circulation (Vandegriff et al., 2003).
Recently, a reductive alkylation chemistry mediated hexaPEGylated Hb, (Propyl-PEG5K)6-Hb, was generated (Hu et al., 2005). The oxygen affinity of (Propyl-PEG5K)6-Hb is comparable to that of (SP-PEG5K)6-Hb, even though Cys-93(β) was unmodified in (Propyl-PEG5K)6-Hb. However, the COP of (Propyl-PEG5K)6-Hb were considerably higher than that of (SP-PEG5K)6-Hb. In general, the COP of the protein solution is a correlate of the number of particles (molecules) in the solution. Accordingly, the higher COP of (Propyl-PEG5K)6-Hb could be a consequence of larger number of molecules in the solution than that in the solution of (SP-PEG5K)6-Hb at the same protein concentration. Typically, Hb undergoes the tetramer-dimer dissociation, which involves cleavage of the non-covalent interactions along the symmetric interfaces α1β2 and α2β1 (Perutz, 1970; Baldwin and Chothia, 1979). Therefore, the different COP values between the two PEGylated proteins are possibly due to the fact that the tetramer-dimer dissociation of Hb is enhanced by the two PEGylation protocols at different levels. Besides, if (Propyl-PEG5K)6-Hb is predominantly present in dimers, it can lead to the high oxygen affinity of (Propyl-PEG5K)6-Hb.
Based on the above, it would be desirable to have a PEGylated hemoglobin with low oxygen affinity. The present invention addresses that need.